基因leg1(liver-enriched gene 1)首先在斑马鱼中作为肝脏富集表达基因被鉴定。进一步的研究揭示leg1编码的Leg1蛋白代表一类新型外分泌蛋白, 它在斑马鱼胚期肝脏生长发育过程中起关键作用。小鼠leg1(mu-leg1)是斑马鱼leg1(zb-leg1)的直系同源基因, 二者编码的蛋白氨基酸序列相似性为31%。文章通过巢式PCR从成年小鼠肝脏中成功克隆了mu-leg1的cDNA序列, 并对该基因在成年小鼠不同组织中的表达特征进行分析和鉴定。Northern印迹杂交和半定量RT-PCR分析结果显示, mu-leg1在成年小鼠小肠中而非肝脏中富集表达。此外, 用制备的mu-Leg1多克隆抗体进行Western印迹杂交, 结果显示mu-Leg1也是一个分泌蛋白。同时, 还建立了mu-leg1基因条件性剔除杂合子小鼠。这些材料为今后深入研究和探讨mu-Leg1蛋白的生化功能奠定了基础。
Gene leg1 (liver-enriched gene 1) was first identified as a novel gene whose expression was enriched in the liver of zebrafish. Further studies revealed that Leg1 protein was a novel secretory protein, which played a role in the liver development in zebrafish. Here we reported the analysis of expression pattern of zb-leg1 homologus gene mu-leg1. The cDNA of mu-leg1 was isolated from adult mouse liver by nested PCR. This gene encodes a putative protein, mu-Leg1, which shares 31% similarity with zb-Leg1 of zebrafish. Both Northern blotting and semi-quantitative RT-PCR demonstrated that the expression of mu-leg1 was enriched in the small intestine rather than in the liver in adult mouse. We also produced a recombined mu-Leg1 protein and a mu-Leg1 specific antibody. West-ern blottingdemonstrated that mu-Leg1 was a secretory protein. In addition, we have established a mu-leg1 condi-tional knock-out heterozygous mouse. Our work builds a basis for further studies of mu-leg1.
[1] Duncan SA. Mechanisms controlling early development of the liver. Mech Dev, 2003, 120(1): 19-33.
[2] Niu XB, Shi H, Peng JR. The role of mesodermal signals during liver organogenesis in zebrafish. Sci China Life Sci, 2010, 53(4): 455-461.
[3] Tao T, Peng JR. Liver development in zebrafish (Danio rerio). J Genet Genomics, 2009, 36(6): 325-334.
[4] Zaret KS. Regulatory phases of early liver development: paradigms of organogenesis. Nat Rev Genet, 2002, 3(7): 499-512.
[5] Cheng W, Guo L, Zhang ZH, Soo HM, Wen CM, Wu W, Peng JR. HNF factors form a network to regulate liver- enriched genes in zebrafish. Dev Biol, 2006, 294(2): 482-496.
[6] Chang CQ, Hu MJ, Zhu ZH, Lo LJ, Chen J, Peng JR, Wang HM. liver-enriched gene 1a and 1b encode novel secretory proteins essential for normal liver development in zebrafish. PLoS ONE, 2011, 6(8): e22910.
[7] Wen CM, Zhang ZH, Ma WP, Xu M, Wen ZL, Peng JR. Genome-wide identification of female- enriched genes in zebrafish. Dev Dyn, 2005, 232(1): 171-179.
[8] Liu PT, Jenkinsinitial NA, Copeland NG. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res, 2003, 13(3): 476-484.
[9] Chan W, Constantino N, Lee SC, Su Q, Melvin D, Court DL, Liu P. A recombineering based approach for high-throughput conditional knockout targeting vector con-struction. Nucleic Acids Res, 2007, 35(8): e64.
[10] Jnyner AL. Gene Targeting. Oxford: Oxford University Press, 1994.
[11] Nagy A. manipulating the mouse embryo: A laboratory manual. New York: Cold Spring Harbor Laboratory Press, 2003: 469, 198, 487.
[12] Aburomia R, Khaner O, Sidow A. Functional evolution in the ancestral lineage of vertebrates or when genomic complexity was wagging its morphological tail. J Struct Funct Genomics, 2003, 3(1-4): 45-52.
[13] Hughes AL. Phylogeny of the Picornaviridae and differential evolutionary divergence of picornavirus proteins. Infect Genet Evol, 2004, 4(2): 143-152.
[14] Otto SP, Whitton J. Polyploid incidence and evolution. Annu Rev Genet, 2000, 34: 401-437.
[15] Nickel W. Unconventional secretory routes: direct protein export across the plasma membrane of mammalian cells. Traffic, 2005, 6(8): 607-614.
[16] Palade G. Intracellular aspects of the process of protein synthesis. Science, 1975, 189(4206): 867.
[17] Stoorvogel W, Kleijmeer MJ, Geuze HJ, Raposo G. The biogenesis and functions of exosomes. Traffic, 2002, 3(5): 321-330.
[18] Kos CH. Cre/loxP system for generating tissue-specific knockout mouse models. Nutr Rev, 2004, 62(6 Pt 1): 243-246.
[19] Brüning JC, Kahn CR, Krone W, Müller-Wieland D. Con-ditional mutagenesis--second generation knockout mice as models for internal diseases. Med Klin(Munich), 1999, 94(10): 564-569.
